The long-term aim of my research program is to develop a quantitative understanding of gene regulation in vivo. This project focuses on the earliest steps in gene regulation, namely, how gene regulatory proteins gain access to their DMAtarget sites in chromatin. Access to target sites inside nucleosomes can occur spontaneously, and can be catalyzed by ATP-dependent nucleosome remodeling factors. We are studying both of these mechanisms. Studies in Aim 1will characterize the spontaneous invasion of nucleosomesby site-specific DMAbinding proteins. We will investigate the kinetics for spontaneous access at interior sites in nucleosomes, the effects of higher order chromatin folding on site accessibility, and the rules governing cooperative binding to nucleosomes. Studies in Aim 2 will address how the Drosophila ISWI/Acf1 nucleosome remodeling machine functions as a machine to catalyze and drive the movement of nucleosomes from one position on DNA to another. We will characterize the protein domain organization of this machine, and the interactions between parts of the machine with each other, and between the machine and the ATP and chromatin substrates. We will characterize how the machine changes conformation through a catalytic cycle, and the changes that the machine induces in nucleosomes. Finally, structural changes taking place within the machine throughout a catalytic cycle will be correlated in time, with those in the nucleosome, to develop a detailed picture of the functioning of this broad class of machine. Relevance: The proper regulation of genes is essential for the development and health of all organisms. Understanding how regulatory proteins find and bind to their DNA target sites will lead, over the long term, to new diagnostics and therapeutics. Studies of ATP-dependent nucleosome remodeling factors have a specific relevance to human development and health, as mutations in these factors are responsible for a wide range of human developmental diseases and cancers, including: Williams syndrome, Schimke immunoosseous dysplasia, Cockayne syndrome, X-linked a thalassaemia, mental retardation, malignant rhabdoid tumors, chronic myeloid leukemia, and prostate and lung carcinomas.